Human development depends on Vascular Endothelial Growth Factor Receptor (VEGFR) signaling, which regulates survival, migration, and proliferation of endothelial cells ? the primary component of the vasculature. Impairment of VEGFR signaling is a main cause of infant deaths due to congenital malformations and decreased birth weight. VEGFR signaling inhibition leads to nephropathy, preeclampsia, and hypertension, while pathological activation is a hallmark of cancer, arterial-venous malformations, and macular degeneration. Since some drugs and industrial chemicals can affect VEGFR signaling, the ability to identify such substances is of primary importance for the protection of human health. However, currently, there is no efficient toxicity screening system employing physiologically relevant cellular models for this purpose. We propose to commercialize a novel high-throughput (HT) platform enabling evaluation of inhibition of endothelial VEGFR- dependent functions using human endothelial colony forming cells (ECFCs). Our proprietary cell growth media VecstemTM allows efficient isolation, robust expansion, and long-term cryopreservation of ECFCs. Thus, we have established ~100 donor-specific ECFCs, which we will commercialize as a unique model of endothelial function. In the proposed product, we have integrated endothelial VEGFR-dependent cytotoxicity, tube formation, and 3D migration assays into a single platform, allowing assessment of a variety of functions important to vascular health. Our cytotoxicity assay is more specific than commercial analogues. Our tube formation assay has 4-times more throughput and 5-times shorter than similar commercial products. Our 3D migration assay has no commercial analogues. In Phase I, we will test the feasibility of our assay and benchmark it against other commercial assays by comparing the responses of our ECFC lines to commercial endothelial cells (e.g., HUVECs) and human fibroblasts (negative control cells), using 20 anti-angiogenic chemicals including known inhibitors of VEGFR signaling. In Aim 1, we will demonstrate the effect of anti- angiogenic chemicals on VEGFR-dependent viability and proliferation of ECFCs. In Aim 2, we will demonstrate the effect of anti-angiogenic chemicals on VEGFR-dependent tube formation and 3D migration of ECFCs. Metrics evaluated will include concentration-response profiles, 50% inhibitory concentration (IC50) values, and the coefficient of variation (CV). The success of this study will justify progression to Phase II, where we will develop and validate a 1536-well HT high content imaging assay protocol using ~1000 chemicals (identified in silico as potentially anti-angiogenic) in several ECFC lines, corroborating the results in a zebrafish anti- angiogenesis assay. We plan to (a) provide a screening service to regulators and other businesses, (b) license our platform to drug discovery companies, and (c) make available stand-alone commercial product(s). Our assay platform has an excellent commercial perspective as U.S. and European regulatory agencies are insisting on the use of alternative methods that replace/reduce the use of animals in pre-clinical toxicology and drug development.